EP0750626A1 - Teilchen von stannius-protein, stanniocalcin - Google Patents

Teilchen von stannius-protein, stanniocalcin

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Publication number
EP0750626A1
EP0750626A1 EP94923154A EP94923154A EP0750626A1 EP 0750626 A1 EP0750626 A1 EP 0750626A1 EP 94923154 A EP94923154 A EP 94923154A EP 94923154 A EP94923154 A EP 94923154A EP 0750626 A1 EP0750626 A1 EP 0750626A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
stannius
dna
corpuscles
Prior art date
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Granted
Application number
EP94923154A
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English (en)
French (fr)
Other versions
EP0750626A4 (de
EP0750626B1 (de
Inventor
Henrik Olsen
Mark D. Adams
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication of EP0750626A1 publication Critical patent/EP0750626A1/de
Publication of EP0750626A4 publication Critical patent/EP0750626A4/de
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Publication of EP0750626B1 publication Critical patent/EP0750626B1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/834—Urine; urinary system
    • Y10S530/835—Kidney

Definitions

  • This invention relates to newly identified polynucleotides, polypeptide ⁇ encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptide ⁇ . More particularly, the polypeptide of the present invention is human Corpuscles of Stannius protein. The invention also relates to inhibiting the action of such polypeptide.
  • Stanniocalcin (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the Corpuscles of Stannius, endocrine glands which are confined to bony fishes.
  • the polypeptide of the present invention has a high degree of homology at the amino acid level to the glycoprotein hormone from fish which is involved in the regulation of calcium levels.
  • the Corpuscles of Stannius protein of non-humans has been studied extensively. Recently, a Corpuscles of Stannius protein has been purified and cloned from Anguilla au ⁇ tralis. The kidneys of teleost fish have been found to contain secretory granules, the Corpuscle ⁇ of Stannius. Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function (Butkus, A. et al. Mol. Cell Endocrinol, 54:123-33 (1987)).
  • hypocalcin the major hypocalcemic hormone of fish.
  • This product is present in relatively large amounts in the Corpuscles of Stannius of several species (i.e., European eel, tilapia goldfish, and carp).
  • Hypocalcin is typically released from the Corpu ⁇ cle ⁇ of Stanniu ⁇ in re ⁇ ponse to an experimentally induced increase of the blood calcium concentration.
  • Ultrastructural observations show that after this treatment the hypocalcin-producing cell type of the corpuscles of stannius are almost completely degranulated.
  • the isolated glycoprotein has an apparent molecular weight of 54 Kda. (Lafeber F. P. et al. , Gen Comp. Endocrinol, 69:19-30 (1988) ) .
  • SUBSTJTUTESHEET(RULE26 stanniocalcin mRNA in salmon is approximately 2 Kda in length and encodes a primary translation product of 256 amino acids. The first 33 residues compri ⁇ e the preprotein region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. (Wagner G. F. et al . , Mol. Cell Endocrinol, 90:7-15 (1992)).
  • a novel mature polypeptide which is a Corpuscles of Stannius protein, as well as fragments, analogs and derivatives thereof.
  • the Corpuscles of Stannius protein of the present invention is of human origin.
  • polynucleotides (DNA or R A) which encode such polypeptide ⁇ .
  • a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes for example, treatment of electrolyte disorders which lead to renal, bone and heart diseases, and disorders due to elevated bone resorption, e.g. osteoporosis and Paget's disease.
  • an antibody against such polypeptide may be used in the treatment of hypocalcemia resulting in tetany, convulsions and other related disorders .
  • Hypocalcemia can arise from a number of different causes including renal failure, hyperparathyroidism, severe infections or burns which traps calcium from the intercellular fluid and from pancreatic insufficiency.
  • antagonist/inhibitors to such polypeptide which may be used to inhibit the action of such polypeptide, for example, in the treatment of hypocalcemia and osteoporosi .
  • FIG. 1 displays the cDNA sequence and corresponding deduced amino acid sequence of the preprocessed Corpuscles of Stannius protein.
  • the amino acid sequence is ⁇ hown below using the standard 3 letter abbreviation code.
  • FIG. 2 displays the comparison of the polypeptide sequence of the human ⁇ tanniocalcin (upper line) to the stanniocalcin isolated from Oncorhynchus kisutch (lower line). No apparent sequence homology exist ⁇ beyond amino acid 204 of the ⁇ tanniocalcin from Oncorhynchus kisutch. Total length of stanniocalcin isolated from Oncorhynchus ki ⁇ utch i ⁇ 256 amino acid ⁇ .
  • FIG. 3 shows the banding pattern of the human stanniocalcin polypeptide following bacterial expression and purification.
  • an isolated nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1, or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75652 on January 25, 1994.
  • the polynucleotide sequence of Figure 1 contains a presequence consisting of the first 15 nucleotides .
  • the polynucleotide sequence encodes a translation product of 247 amino acids of which the first 33 amino acids may represent a prepro region of the protein. Thus, cleavage of the prepro region results in a mature active polypeptide of 214 amino acids.
  • the polynucleotide of the present invention wa ⁇ isolated from a human early stage lung cDNA library. It contains an open reading frame encoding a prepropolypeptide of 247 amino acids.
  • the polypeptide has a high degree of homology to Stanniocalcin from Anguilla australi ⁇ , 119 identical amino acids (61%) in a 195 amino acid overlap. It also has a very high homology to stanniocalcin from Oncorhynchus kisutch, 118 identical amino acids (57%) in a 204 amino acid overlap.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single- ⁇ tranded and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the depo ⁇ ited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3 ' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of ⁇ uch polynucleotides which variant ⁇ encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variant ⁇ , substitution variants and addition or insertion variant ⁇ .
  • the polynucleotide may have a coding ⁇ equence which i ⁇ a naturally occurring allelic variant of the coding ⁇ equence shown in Figure 1 or of the coding sequence of the deposited clone.
  • the present invention also includes polynucleotides wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions a ⁇ a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5 ' amino acid re ⁇ idues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains .
  • the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a pre ⁇ equence (leader sequence).
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allow ⁇ for purification of the polypeptide of the pre ⁇ ent invention.
  • the marker ⁇ equence may be a hexa- hi ⁇ tidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
  • the deposit(s) referred to herein will be maintained under the Budape ⁇ t Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are provided merely as a convenience and are not an admission that a deposit i ⁇ required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotides contained in the deposited materials, as well a ⁇ the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein.
  • a license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
  • the present invention further relates to a Corpuscles of Stannius polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of ⁇ uch polypeptide.
  • fragment when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retain ⁇ e ⁇ sentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residue ⁇ are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and ⁇ uch substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residue ⁇ includes a sub ⁇ tituent group, or (iii) one in which the mature polypeptide i ⁇ fused with another compound, such a ⁇ a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein ⁇ equence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • i ⁇ olated that the material i ⁇ removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide pre ⁇ ent in a living animal i ⁇ not i ⁇ olated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that ⁇ uch vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the Corpuscle ⁇ of Stannius genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing a polypeptide by recombinant techniques.
  • the polynucleotide sequence may be included in any one of a variety of expre ⁇ sion vehicles, in particular vector ⁇ or pla ⁇ mid ⁇ for expre ⁇ ing a polypeptide.
  • vector ⁇ include chromo ⁇ omal, nonchromo ⁇ omal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmid ⁇ ; phage DNA; yeast plasmids; vector ⁇ derived from combination ⁇ of pla ⁇ mid ⁇ and phage DNA, viral DNA such as vaccinia, adenoviru ⁇ , fowl pox viru ⁇ , and pseudorabies.
  • any other plasmid or vector may be u ⁇ ed as long as they are replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence i ⁇ in ⁇ erted into an appropriate re ⁇ triction endonuclea ⁇ e ⁇ ite( ⁇ ) by procedure ⁇ known in the art.
  • procedure ⁇ and other ⁇ are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expres ⁇ ion vector i ⁇ operatively linked to an appropriate expression control sequence( ⁇ ) (promoter) to direct mRNA synthesis.
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expres ⁇ ion of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expres ⁇ ion vector ⁇ preferably contain a gene to provide a phenotypic trait for selection of transformed ho ⁇ t cell ⁇ ⁇ uch a ⁇ dihydrofolate reducta ⁇ e or neomycin re ⁇ istance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence a ⁇ hereinabove de ⁇ cribed, a ⁇ well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as a ⁇ E. coli. Salmonella typhimurium; Streptomyce ⁇ ; fungal cell ⁇ , ⁇ uch a ⁇ yea ⁇ t; insect cells, ⁇ uch a ⁇ Dro ⁇ ophila or Sf9; animal cell ⁇ such as CHO, COS or Bowes melanoma; plant cell ⁇ , etc.
  • Salmonella typhimurium Salmonella typhimurium
  • Streptomyce ⁇ typhimurium
  • fungal cell ⁇ ⁇ uch a ⁇ yea ⁇ t
  • insect cells ⁇ uch a ⁇ Dro ⁇ ophila or Sf9
  • animal cell ⁇ such as CHO, COS or Bowes melanoma
  • plant cell ⁇ etc.
  • the pre ⁇ ent invention al ⁇ o include ⁇ recombinant con ⁇ truct ⁇ compri ⁇ ing one or more of the ⁇ equence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the constructs comprise a vector, ⁇ uch a ⁇ a pla ⁇ mid or viral vector, into which a ⁇ equence of the invention ha ⁇ been in ⁇ erted, in a forward or reverse orientation.
  • the con ⁇ truct further comprises regulatory sequence ⁇ , including, for example, a promoter, operably linked to the sequence. Large numbers of ⁇ uitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBs, pDIO, phagescript, P ⁇ iX174, Pbluescript SK, Pbsks, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene) ; pTrc99A, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic PWLNEO, pSV2cat, pOG44, pXTl, PSG (Stratagene) pSVK3, PBPV, PMSG, PSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transfera ⁇ e) vector ⁇ or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kina ⁇ e, early and late SV40, LTR ⁇ from retroviru ⁇ , and mouse metallothionein-I . Selection of the appropriate vector and promoter is well within the level of ordinary ⁇ kill in the art.
  • the pre ⁇ ent invention relates to host cell ⁇ containing the above-de ⁇ cribed con ⁇ truct.
  • the ho ⁇ t cell can be a higher eukaryotic cell, ⁇ uch a ⁇ a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated tran ⁇ fection, or electroporation (Davi ⁇ , L., Dibner, M., Battey, I., Ba ⁇ ic Method ⁇ in Molecular Biology, (1986) ) .
  • the con ⁇ truct ⁇ in host cell ⁇ can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be ⁇ ynthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA con ⁇ tructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclo ⁇ ure of which i ⁇ hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviru ⁇ enhancer ⁇ .
  • recombinant expre ⁇ ion vectors will include origins of replication and ⁇ electable marker ⁇ permitting transformation of the ho ⁇ t cell, e.g., the ampicillin re ⁇ i ⁇ tance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural ⁇ equence.
  • Such promoters can be derived from operons encoding glycolytic enzymes ⁇ uch a ⁇ 3-phosphoglycerate kinase (PGK), ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is as ⁇ embled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteri ⁇ tic ⁇ , e.g., ⁇ tabilization or ⁇ implified purification of expre ⁇ sed recombinant product.
  • Useful expres ⁇ ion vector ⁇ for bacterial u ⁇ e are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to en ⁇ ure maintenance of the vector and to, if de ⁇ irable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various specie ⁇ within the genera Pseudomona ⁇ , Streptomyce ⁇ , and Staphylococcus, although others may al ⁇ o be employed as a matter of choice.
  • useful expression vector ⁇ for bacterial use can comprise a ⁇ electable marker and bacterial origin of replication derived from commercially available plasmid ⁇ compri ⁇ ing genetic element ⁇ of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vector ⁇ include, for example, pKK223-3 (Pharmacia Fine Chemical ⁇ , Upp ⁇ ala, Sweden) and GEM1 (Promega Biotec, Madi ⁇ on, WI, USA).
  • pBR322 "backbone" section ⁇ are combined with an appropriate promoter and the ⁇ tructural ⁇ equence to be expre ⁇ ed.
  • the selected promoter is induced by appropriate means (e.g., temperature ⁇ hift or chemical induction) and cell ⁇ are cultured for an additional period.
  • appropriate means e.g., temperature ⁇ hift or chemical induction
  • Cell ⁇ are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture system ⁇ can also be employed to express recombinant protein.
  • mammalian expression system ⁇ include the COS-7 line ⁇ of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expres ⁇ ion vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, ⁇ plice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed ⁇ equence ⁇ .
  • DNA ⁇ equence ⁇ derived from the SV40 ⁇ plice, and polyadenylation ⁇ ite ⁇ may be u ⁇ ed to provide the required nontran ⁇ cribed genetic elements.
  • HPLC high performance liquid chromatography
  • the polypeptide ⁇ of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cell ⁇ in culture). Depending upon the ho ⁇ t employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptide ⁇ of the invention may al ⁇ o include an initial methionine amino acid re ⁇ idue.
  • the polypeptide of the pre ⁇ ent invention may be u ⁇ ed for therapeutic treatment of numerous electrolyte-based diseases.
  • One cause of arterial hypertension is abnormal Na + transport acros ⁇ the cell wall of the vascular smooth cells due to a defect in or inhibition of the Na-K pump, another is increased permeability to Na + as has been described in some for ⁇ of human hypertension.
  • the net result is increases in intracellular Na + , which makes the cell more sensitive to vasoconstrictive agent ⁇ .
  • Ca ++ follows Na + , it is postulated that it is the accumulation of intracellular Ca ++ and not Na + per ⁇ e that is responsible for increased sen ⁇ itivity to sympathetic stimulation.
  • Corpuscle ⁇ of Stanniu ⁇ protein can function a ⁇ a hypocalcemic agent it can help to off ⁇ et this increased intracellular Ca ++ and reduce or prevent hypertension. Further, hypercalcemia has been implicated in heart dysrhythmia ⁇ , coma and cardiac arre ⁇ t. Accordingly, the Corpu ⁇ cle ⁇ of Stannius protein may have therapeutic value for the treatment of these disorders by lowering the concentration of free calcium.
  • Hypertension is also directly related to renal disorder ⁇ . Accordingly, a higher or lower than normal concentration of electrolyte ⁇ can cau ⁇ e renal malfunction and directly lead to other more ⁇ eriou ⁇ di ⁇ orders .
  • calcium-phosphoru ⁇ imbalance can cau ⁇ e mu ⁇ cle and bone pain, demineralization of the bones and calcification in variou ⁇ organ ⁇ including the brain, eyes, myocardium and blood vessels.
  • the polypeptide of the present invention may be used to offset disorders that are due to a calcium-phosphate imbalance. Renal failure it ⁇ elf leads to an abnormally high concentration of phosphate in the blood which can be reduced to normal concentration ⁇ by the polypeptide of the pre ⁇ ent invention.
  • the polypeptide of the pre ⁇ ent invention is also useful for the treatment of certain bone disea ⁇ e ⁇ . For example, excessive concentrations of calcium lead to the development of fibrous nodules in affected bone.
  • the causes of hypercalcemia may also be a number of different disorder ⁇ including hyperparathyroidism, hypervitaminosis D, tumors that raises the serum calcium levels by destroying bone, ⁇ arcoidosis, hyperthyroidism, adrenal in ⁇ ufficiency, loss of serum albumin secondary to renal diseases, exces ⁇ ive GI calcium ab ⁇ orption and elevated concentration of pla ⁇ ma protein ⁇ . Accordingly, Corpu ⁇ cles of Stannius protein is effective in reducing hypercalcemia and its related disorder ⁇ .
  • Corpuscle ⁇ of Stanniu ⁇ protein may also be useful for the treatment of other disorder ⁇ relating to unu ⁇ ual electrolyte concentrations and fluid imbalance, for example, migraine headaches.
  • polypeptide may also be employed in accordance with the present invention by expression of such polypeptide in vivo , which is often referred to a ⁇ "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding the polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding the polypeptide of the present invention.
  • cells may be engineered in vivo for expression of the polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expres ⁇ ion of the polypeptide in vivo .
  • the expre ⁇ ion vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • the polypeptides of the present invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • a ⁇ ociated with ⁇ uch container( ⁇ ) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, u ⁇ e or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or ⁇ ale for human admini ⁇ tration.
  • the polypeptide of the pre ⁇ ent invention may be employed in conjunction with other therapeutic compound ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ may be admini ⁇ tered in a convenient manner ⁇ uch a ⁇ by the oral, intravenou ⁇ , intraperitoneal, intramu ⁇ cular, ⁇ ubcutaneou ⁇ , intrana ⁇ al, or intrader al route ⁇ .
  • the amounts and dosage regimens administered to a subject will depend on a number of factors such as the mode of admini ⁇ tration, the nature of the condition being treated, the body weight of the ⁇ ubject being treated and the judgment of the pre ⁇ cribing phy ⁇ ician.
  • do ⁇ e ⁇ of at lea ⁇ t about 0.1 mg/kg body weight and in mo ⁇ t cases will not be administered in an amount in exces ⁇ of about 10.0 mg/kg body weight, and preferably i ⁇ given in do ⁇ e ⁇ of about 0.1 mg/kg body weight to about 1.0 mg/kg body weight daily, for several days, taking into account the routes of administration, symptom ⁇ , etc.
  • the ⁇ equence ⁇ of the pre ⁇ ent invention are al ⁇ o valuable for chromosome identification.
  • the ⁇ equence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromo ⁇ ome marking reagent ⁇ ba ⁇ ed on actual ⁇ equence data (re- at polymorphisms) are presently available for marking chr -r.o ⁇ omal location.
  • the mapping of DNA ⁇ to chromosomes according to the present invention is an important first step in correlating those ⁇ equence ⁇ with genes a ⁇ ociated with di ⁇ ea ⁇ e.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
  • Computer analysis of the cDNA is used to rapidly select primers that do not ⁇ pan more than one exon in the genomic DNA, thus complicating the amplification proces ⁇ .
  • These primer ⁇ are then u ⁇ ed for PCR screening of somatic cell hybrids containing individual human chromosome ⁇ . Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids is a rapid procedure for as ⁇ igning a particular DNA to a particular chromo ⁇ ome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategie ⁇ that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosome ⁇ and pre ⁇ election by hybridization to construct chromosome specific-cDNA libraries .
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient ⁇ ignal intensity for simple detection.
  • FISH require ⁇ use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 i ⁇ probably not necessary to get good results a reasonable percentage of the time.
  • the phy ⁇ ical po ⁇ ition of the ⁇ equence on the chromo ⁇ ome can be correlated with genetic map data.
  • genetic map data are found, for example, in V. McKu ⁇ ick, Mendelian Inheritance in Man (available on line through John ⁇ Hopkin ⁇ University Welch Medical Library) .
  • the relationship between genes and disease ⁇ that have been mapped to the same chromo ⁇ omal region are then identified through linkage analy ⁇ is (coinheritance of physically adjacent gene ⁇ ).
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative gene ⁇ . (Thi ⁇ assume ⁇ 1 megabase mapping resolution and one gene per 20 kb) .
  • Comparison of affected and unaffected individuals generally involves fir ⁇ t looking for ⁇ tructural alteration ⁇ in the chromosomes, such as deletions or translocations that are visible from chromosome spread ⁇ or detectable u ⁇ ing PCR ba ⁇ ed on that cDNA ⁇ equence.
  • complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms .
  • the present invention is further directed to inhibiting Corpuscles of Stannius protein in vivo by use of antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide ⁇ equence, which encodes for the mature polypeptide of the pre ⁇ ent invention, i ⁇ used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in tran ⁇ cription (triple helix - see Lee et al. , Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al. , Science, 251: 1360 (1991)), thereby preventing transcription and the production of Corpuscle ⁇ of Stanniu ⁇ protein.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the Corpuscles of Stannius protein (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides a ⁇ Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) .
  • the oligonucleotides described above can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo to inhibit production of Corpuscles of Stannius protein in the manner described above.
  • antisense constructs of Corpuscles of Stannius protein can be used to treat hypocalcemia by inactivating Corpuscle ⁇ of Stanniu ⁇ protein and therefore preventing a hypocalcemic effect.
  • Osteoporosis characterized by thin and brittle bones due to a low level of calcium, can be treated by the above-described antisen ⁇ e constructs.
  • these antisense construct ⁇ may be used to treat Paget's disease.
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expre ⁇ sing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, CDR grafted single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expres ⁇ ion library. Variou ⁇ procedures known in the art may be used for the production of such antibodies and fragments .
  • Antibodies generated against the polypeptide corresponding to a sequence of the present invention or its in vivo receptor can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide it ⁇ elf. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tis ⁇ ue expre ⁇ ing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuou ⁇ cell line culture ⁇ can be u ⁇ ed.
  • Example ⁇ include the hybridoma technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodie ⁇ (Cole, et al. , 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Li ⁇ , Inc., pp. 77- 96).
  • Antibodie ⁇ specific to the polypeptide of the present invention may further be u ⁇ ed to inhibit the proper functioning of the polypeptide.
  • the antibodies may be u ⁇ ed a ⁇ antihypocalcemic agents by preventing the hypocalcemic function of the protein of ⁇ tanniocalcin.
  • diseases which may be treated with these antibodie ⁇ i ⁇ osteoporosis.
  • the present invention i ⁇ al ⁇ o directed to antagoni ⁇ t/inhibitor ⁇ of the polypeptide ⁇ of the pre ⁇ ent invention.
  • the antagoni ⁇ t/inhibitors are those which inhibit or eliminate the function of the polypeptide.
  • antagonist ⁇ can bind to a polypeptide of the pre ⁇ ent invention and inhibit or eliminate its function.
  • the antagonist for example, could be an antibody against the polypeptide which binds to the polypeptide or, in some cases, an oligonucleotide.
  • An example of an inhibitor is a small molecule which binds to the catalytic site of the polypeptide thereby making it inaccessible to substrate and preventing biological activity. Examples, of small molecule inhibitors include but are not limited to small peptides or peptide-like molecules.
  • antagonists to the polypeptides of the present invention may be employed which bind to the receptors to which a polypeptide of the present invention normally binds.
  • the antagonists may be closely related proteins such that they recognize and bind to the receptor site ⁇ of the natural protein, however, they are inactive form ⁇ of the natural protein and thereby prevent the action of Corpu ⁇ cle ⁇ of Stanniu ⁇ protein, since receptor sites are occupied. In these ways, the action of the Corpuscles of Stannius protein is prevented and the antagonist/inhibitors may be used as anti-hypocalcemic agents, to treat osteoporo ⁇ i ⁇ among other di ⁇ order ⁇ where an increase in calcium levels is desired.
  • the antagonist/inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.
  • “Pla ⁇ mids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers .
  • the ⁇ tarting pla ⁇ mids herein are either commercially available, publicly available on an unrestricted basi ⁇ , or can be con ⁇ tructed from available pla ⁇ mid ⁇ in accord with published procedures.
  • equivalent plasmid ⁇ to tho ⁇ e de ⁇ cribed are known in the art and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • “Dige ⁇ tion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain ⁇ equences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment i ⁇ u ⁇ ed with about 2 unit ⁇ of enzyme in about 20 ⁇ l of buffer ⁇ olution.
  • buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 °C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Oligonucleotides refers to either a ⁇ ingle stranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trands which may be chemically synthe ⁇ ized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A ⁇ ynthetic oligonucleotide will ligate to a fragment that ha ⁇ not been depho ⁇ phorylated.
  • Ligase refers ⁇ to the process of forming phosphodie ⁇ ter bond ⁇ between two double ⁇ tranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amount ⁇ of the DNA fragment ⁇ to be ligated.
  • ligase T4 DNA ligase
  • the DNA sequence encoding for Corpuscles of Stannius Protein (ATCC # 75652) is initially amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of Corpuscle ⁇ of Stanniu ⁇ Protein and additional nucleotide ⁇ corre ⁇ ponding to SphI restriction site and Bgll were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer has the sequence 5'- GACTGCATGCTCCAAAACTCAGCAGTG-3' , contains a SphI re ⁇ triction enzyme ⁇ ite (GCATGC) and 21 nucleotide ⁇ of Corpu ⁇ cle ⁇ of Stanniu ⁇ Protein coding sequence starting from the initiation codon;
  • GCATGC SphI re ⁇ triction enzyme ⁇ ite
  • the 3' sequence 3 '-GACTAGATCTTGCACTCTCATGGGATGTGCG-5' contains complementary sequences to Bgl II restriction site (AGATCT) and the last 21 nucleotides of Corpu ⁇ cle ⁇ of Stanniu ⁇ Protein coding sequence.
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expres ⁇ ion vector pQE70 (Qiagen, Inc.
  • pQE70 encode ⁇ antibiotic re ⁇ i ⁇ tance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/0), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites.
  • pQE70 was then digested with SphI and Bgl II restriction enzymes.
  • the amplified ⁇ equence ⁇ were ligated into pQE70 and were in ⁇ erted in frame with the ⁇ equence encoding for the hi ⁇ tidine tag and the RBS.
  • the ligation mixture wa ⁇ then u ⁇ ed to tran ⁇ form E.
  • M15/rep4 available from Qiagen under the trademark M15/rep 4.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repres ⁇ or and also confers kana ycin resi ⁇ tance (Kan r ). Transformants are identified by their ability to grow on LB plates and a picillin/kanamycin resi ⁇ tant colonie ⁇ were selected. Plasmid DNA was isolated and confirmed by restriction analysi ⁇ . Clone ⁇ containing the de ⁇ ired con ⁇ truct ⁇ were grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • Tho O/N culture is u ⁇ ed to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cell ⁇ were grown to an optical den ⁇ ity 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induce ⁇ by inactivating the lad repre ⁇ or, clearing the P/O leading to increased gene expres ⁇ ion.
  • Cell ⁇ were grown an extra 3 to 4 hour ⁇ . Cells were then harvested by centrifugation (20 mins at 6000Xg) . The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • solubilized ⁇ tanniocalcin wa ⁇ purified from this solution by chromatography on a Nickel-Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-His tag.
  • solubilized ⁇ tanniocalcin wa ⁇ purified from this solution by chromatography on a Nickel-Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-His tag.
  • Protein renaturation out of GnHCl can be accompli ⁇ hed by ⁇ everal protocols.
  • step dialysis is utilized to remove the GnHCL.
  • the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decrea ⁇ ing linear GnHCL gradient i ⁇ run.
  • the protein i ⁇ allowed to renature while bound to the column and i ⁇ subsequently eluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol.
  • soluble protein is dialyzed again ⁇ t a storage buffer containing 5 mM Ammonium Bicarbonate.
  • the purified protein was analyzed by SDS-PAGE. See Figure 3.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • 150 155 160 lie Leu Gin Thr Asp Hi ⁇ Cys Ala Gin Thr Hi ⁇ Pro Arg Ala A ⁇ p

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US6008322A (en) * 1996-04-02 1999-12-28 Zymogenetics, Inc. Stanniocalcin-2
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US6902890B1 (en) 1999-11-04 2005-06-07 Diadexus, Inc. Method of diagnosing monitoring, staging, imaging and treating cancer
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US20040009171A1 (en) * 2001-10-18 2004-01-15 Genentech, Inc. Methods for the treatment of carcinoma
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US7056685B1 (en) 2002-11-05 2006-06-06 Amgen Inc. Receptor ligands and methods of modulating receptors
US8759298B2 (en) 2010-05-03 2014-06-24 Scott & White Healthcare Protein therapy for corneal inflammation, epithelial wound healing, and photoreceptor degeneration

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